Actin dynamics in the regulation of the function of hematopoietic stem cells

在之前的资助期内，我们发现在wnt5a - haplod不足的小鼠中，移植后造血干细胞再生数量正常。然而，再生的hsc （HSC-5a）存在功能缺陷，不能移植继发受体。为了了解这种功能衰退，我们进行了转录组分析。这一分析表明，HSC-5a中的肌动蛋白动力学被解除调控，这在实验中得到证实，显示出粘附、迁移和归巢减少。然而，这些迁移特性的减少并不能解释移植完全失败。因此，在当前的应用中，我们建议研究肌动蛋白动力学的解除是如何驱动造血干细胞功能下降的。众所周知，肌动蛋白动力学不仅在迁移中需要，而且在胞内和胞外作用以及自噬和有丝分裂中也需要。事实上，我们的初步分析显示HSC-5a中线粒体数量增加，提示线粒体自噬缺陷。在这里，我们将验证HSC-5a在调节线粒体数量的动作蛋白依赖过程中失败的假设。为此，我们将研究功能缺陷造血干细胞的线粒体质量和功能，以及它们与肌动蛋白动力学途径表达失调的关系。这些造血干细胞不仅可以在Wnt5a- haploindeficient小鼠中产生，也可以从Osx-Cre；Wnt5a<s:1>小鼠中分离出来。此外，我们将使用肌动蛋白动力学的小分子抑制剂来调节有丝分裂，或者，我们将有条件地调节HSC-5a中上调的肌动蛋白动力学基因的表达。重要的是，我们将研究这些治疗或基因操作是否会挽救体外行为并在体内重新填充HSC活性。这些实验一方面将增加我们对肌动蛋白动力学、线粒体功能和HSC功能之间耦合关系的理解，并发现可能促进线粒体正常化和HSC功能恢复的其他肌动蛋白动力学通路节点。这一知识不仅对理解造血干细胞在正常再生和衰老过程中的功能下降具有重要意义，而且对再生障碍性贫血、慢性淋巴细胞白血病和骨髓增生异常综合征等与异常生态位相关的恶性疾病也具有重要意义。

In the previous funding period, we have found that in Wnt5a-haploinsufficient mice, HSCs are regenerated in normal numbers after transplantation. However, the regenerated HSCs (HSC-5a) are functionally defective and do not engraft secondary recipients. To understand this functional decline, we performed a transcriptome analysis. This analysis suggested that actin dynamics is deregulated in the HSC-5a, which was confirmed in experiments showing reduced adhesion, migration and homing. However, these diminished migratory properties do not explain the complete failure to engraft. Hence, in the current application, we propose to investigate how deregulation of actin dynamics drives the functional decline of HSCs. It is known that actin dynamics is not only required in migration, but also in endo- and exocytosis, as well as auto- and mitophagy. Indeed, our preliminary analyses showed an increased number of mitochondria in HSC-5a, suggestive of defective mitophagy. Here, we will test the hypothesis that HSC-5a suffer from a failure of an actin-dependent process to regulate the number of mitochondria. For this purpose, we will study mitochondrial mass and function in functionally deficient HSCs and their association with deregulated expression of the actin dynamics pathway. These HSCs will not only be generated in Wnt5a-haploinsuficient mice, but also isolated from Osx-Cre;Wnt5amice. Furthermore, we will modulate mitophagy, using small molecules inhibitors of actin dynamics or, alternatively, we will conditionally modulate the expression of actin dynamics genes which are upregulated in HSC-5a. Importantly, we will study whether these treatments or genetic manipulations will rescue in vitro behavior and repopulating HSC activity in vivo. These experiments will on the one hand increase our understanding of the coupling between actin dynamics, mitochondrial function, and HSC function, as well as identify additional actin dynamics pathway nodes which may facilitate normalization of mitochondria and rescue of HSC function. This knowledge may not only be of importance in understanding the functional decline of HSCs in normal regeneration and aging, but also in malignant disease associated with an aberrant niche such as aplastic anemia, chronic lymphocytic leukemia and myelodysplastic syndrome.